Mitoriboscins: mitochondrial-based therapeutics targeting cancer cells, bacteria, and pathogenic yeast

ABSTRACT

The present disclosure relates to inhibitors of mitochondrial function. Methods of screening compounds for mitochondrial inhibition are disclosed. Also described are methods of using mitochondrial inhibitors called mitoriboscins—mitochondrial-based therapeutic compounds having anti-cancer and antibiotic properties—to prevent or treat cancer, bacterial infections, and pathogenic yeast, as well as methods of using mitochondrial inhibitors to provide anti-aging benefits. Specific mitoriboscin compounds and groups of mitoriboscins are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Application No.PCT/US2018/022403 filed Mar. 14, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/471,688 filed Mar. 15, 2017, theentire contents of each of which are hereby incorporated by reference.

FIELD

The present disclosure relates to novel inhibitors of mitochondrialfunction that target the mitochondrial ribosome, referred to herein as“mitoriboscins,” methods for identifying mitoriboscins, methods of usingthe inhibitors to target cancer stem cells, to target bacteria andpathogenic yeast, and to provide anti-aging benefits, and pharmaceuticalcompositions for treating cancer, bacterial infections, yeastinfections, and aging, containing one or more mitoriboscins as theactive ingredient.

BACKGROUND

Researchers have struggled to develop new anti-cancer treatments.Conventional cancer therapies (e.g. irradiation, alkylating agents suchas cyclophosphamide, and anti-metabolites such as 5-Fluorouracil) haveattempted to selectively detect and eradicate fast-growing cancer cellsby interfering with cellular mechanisms involved in cell growth and DNAreplication. Other cancer therapies have used immunotherapies thatselectively bind mutant tumor antigens on fast-growing cancer cells(e.g., monoclonal antibodies). Unfortunately, tumors often recurfollowing these therapies at the same or different site(s), indicatingthat not all cancer cells have been eradicated. Relapse may be due toinsufficient chemotherapeutic dosage and/or emergence of cancer clonesresistant to therapy. Hence, novel cancer treatment strategies areneeded. Similarly, researchers have struggled to develop new antibiotictreatments. Antibiotic resistance has developed due to gradual buildupof random mutations in the microbes and the misuse of antibiotics. Poorfinancial investment in antibiotic research and development has worsenedthe situation. Hence, novel antibiotic treatment strategies are needed.

Advances in mutational analysis have allowed in-depth study of thegenetic mutations that occur during cancer development. Despite havingknowledge of the genomic landscape, modern oncology has had difficultywith identifying primary driver mutations across cancer subtypes. Theharsh reality appears to be that each patient's tumor is unique, and asingle tumor may contain multiple divergent clone cells. What is needed,then, is a new approach that emphasizes commonalities between differentcancer types. Targeting the metabolic differences between tumor andnormal cells holds promise as a novel cancer treatment strategy. Ananalysis of transcriptional profiling data from human breast cancersamples revealed more than 95 elevated mRNA transcripts associated withmitochondrial biogenesis and/or mitochondrial translation. Sotgia etal., Cell Cycle, 11(23):4390-4401 (2012). Additionally, more than 35 ofthe 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs).Proteomic analysis of human breast cancer stem cells likewise revealedthe significant overexpression of several mitoribosomal proteins as wellas other proteins associated with mitochondrial biogenesis. Lamb et al.,Oncotarget, 5(22):11029-11037 (2014). Functional inhibition ofmitochondrial biogenesis using the off-target effects of certainbacteriostatic antibiotics or OXPHOS inhibitors provides additionalevidence that functional mitochondria are required for the propagationof cancer stem cells.

There exists a need in the art for novel anticancer strategies, newcompounds with broad-spectrum antibiotic activity, and compounds toreduce the effects of aging. The “endo-symbiotic theory of mitochondrialevolution” can be used as the basis for the development of therapies totreat drug-resistance that is characteristic of both tumor recurrenceand infectious disease, and such therapies may have the additionalbenefit of slowing the aging process.

SUMMARY

In view of the foregoing background, it is therefore an object of thisdisclosure to demonstrate that mitochondrial biogenesis plays a criticalrole in the propagation and maintenance of many cancers. It is also anobject of this disclosure to present methods for identifyingmitochondrial inhibitors that bind to the mitochondrial ribosome (largesub-unit or small sub-unit) and have anti-cancer and antibioticproperties. It is also an object of this disclosure to identifymitochondrial inhibitors and groups thereof and having anti-cancer andantibiotic properties. It is also an object of this disclosure toidentify classes of mitochondrial inhibitors having anti-agingproperties. It is also an object of this disclosure to identify classesof mitochondrial inhibitors that function as radiosensitizers andphotosensitizers.

The present disclosure relates to mitochondrial inhibitor compounds thathave anti-cancer and antimicrobial activity, radiosensitizing andphotosensitizing effects, as well as anti-aging effects. The term“mitoriboscins” broadly refers to mitoribosome-targeted therapeuticshaving anti-cancer and antibiotic properties. These compounds bind toeither the large sub-unit or the small sub-unit of the mitoribosome (orin some instances, both) and inhibit mitochondrial biogenesis. Thepresent disclosure further relates to methods of identifyingmitoriboscins, methods of making such mitoriboscins, and methods ofusing mitoriboscins for therapeutic purposes.

The inventors analyzed phenotypic properties of cancer stem cells (CSCs)that could be targeted across a wide range of cancer types, andidentified a strict dependence of CSCs on mitochondrial biogenesis forthe clonal expansion and survival of a CSC. Previous work by theinventors demonstrated that different classes of FDA-approvedantibiotics, and in particular tetracyclines such as doxycycline anderythromycin, have an off-target effect of inhibiting mitochondrialbiogenesis. As a result, such compounds could have efficacy foreradicating CSCs. However, these common antibiotics were not designed totarget the mitoribosome, and therefore have limited anti-cancerproperties. Under the present approach, the inventors have identifiedcompounds that target the mitochondrial ribosome, or mitoribosome, andinhibit mitochondrial biogenesis. These mitoribosome-targetingcompounds—mitoriboscins—therefore have highly potent anticancerproperties, among other advantageous properties.

Given the role of mitochondrial biogenesis in cell propagation,mitochondrial inhibitors as identified under the present approachprovide an entirely new class of cancer therapy. In addition to theirpotential use as cancer therapies, mitoriboscins serve as usefulbroad-spectrum antibiotics. The endo-symbiotic theory of mitochondrialevolution theorizes that mitochondrial organelles evolved from engulfedaerobic bacteria following millions of years of symbiosis andadaptation. The evolutionary history of mitochondrial organellessuggests that compounds that target mitochondrial protein translation incancer cells also possess anti-microbial activity. Indeed, as discussedbelow, mitoriboscins have demonstrated antibiotic properties.

Additionally, studies on genetic inhibition of mitochondrial proteintranslation have shown beneficial “side effects,” such as the slowing ofthe aging process and increased lifespan in model organisms. Theseresults suggest that mitochondrial inhibitors may also be useful foranti-aging therapies, which is the subject of ongoing studies.

Novel mitochondrial inhibitors may be identified through a convergentapproach of virtual high-throughput in silico screening followed by invitro validation for mitochondrial inhibition. New mitochondrialinhibitors can be rapidly developed by combining in silico drug designwith phenotypic drug screening.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the endo-symbiotic theory of mitochondrial evolution.

FIG. 2 shows a schematic diagram outlining a drug discovery strategyaccording to embodiments of the present approach.

FIG. 3 shows the effects of ten candidate mitoriboscin compounds onATP-depletion in MCF7 cells.

FIGS. 4A-4D illustrates the chemical structures of ten mitoriboscincompounds identified following phenotypic drug screening. Thesestructures are grouped into four groups—mitoribocyclines (compounds a-con FIG. 4A), mitoribomycins (compounds d-g on FIG. 4B), mitoribosporins(compound h and i on FIG. 4C), and mitoribofloxins compound j on FIG.4D).

FIG. 5 shows the effects of seven mitoriboscin compounds on mammosphereformation in MCF7 cells.

FIG. 6A shows the effects of three mitoriboscin compounds on the cellviability of MCF7 cells. FIG. 6B shows the effects of three mitoriboscincompounds on the cell viability of hTERT-BJ1 cells.

FIG. 7A shows the effects of compound 23/G4 on oxygen consumption rate(OCR) over time in MCF7 cells. FIG. 7B shows the effects of compound23/G4 on extracellular acidification rate (ECAR) over time in MCF7cells. FIG. 7C shows the effects of compound 23/G4 on OCR for basalrespiration, proton leak, ATP-linked respiration, maximal respiration,and spare respiratory capacity. FIG. 7D shows the effects of compound23/G4 on ECAR for glycolysis, glycolytic reserve, and glycolytic reservecapacity.

FIG. 8A shows the effects of compound 24/D4 on oxygen consumption rate(OCR) over time in MCF7 cells. FIG. 8B shows the effects of compound24/D4 on extracellular acidification rate (ECAR) over time in MCF7cells. FIG. 8C shows the effects of compound 24/D4 on OCR for basalrespiration, proton leak, ATP-linked respiration, maximal respiration,and spare respiratory capacity. FIG. 8D shows the effects of compound24/D4 on ECAR for glycolysis, glycolytic reserve, and glycolytic reservecapacity.

FIG. 9A shows the effects of compound 24/F9 on oxygen consumption rate(OCR) over time in MCF7 cells. FIG. 9B shows the effects of compound24/F9 on extracellular acidification rate (ECAR) over time in MCF7cells. FIG. 9C shows the effects of compound 24/F9 on OCR for basalrespiration, proton leak, ATP-linked respiration, maximal respiration,and spare respiratory capacity. FIG. 9D shows the effects of compound24/F9 on ECAR for glycolysis, glycolytic reserve, and glycolytic reservecapacity.

FIG. 10A shows the effects often mitoriboscin compounds on maximalrespiration in MCF7 cells. FIG. 10B shows the effects of tenmitoriboscin compounds on ATP production in MCF7 cells.

FIG. 11 shows the effects of three mitoriboscin compounds at twodifferent concentrations on cell migration in MDA-MB-231 cells.

FIG. 12 illustrates the four new classes of mitochondrialinhibitors—mitoribocyclines, mitoribomycins, mitoribosporins, andmitoribofloxins.

DESCRIPTION

The following description illustrates embodiments of the presentapproach in sufficient detail to enable practice of the presentapproach. Although the present approach is described with reference tothese specific embodiments, it should be appreciated that the presentapproach can be embodied in different forms, and this description shouldnot be construed as limiting any appended claims to the specificembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the present approach to those skilled in the art.

The mitochondrial ribosome is an untapped gateway for treating a numberof afflictions, ranging from cancer to bacterial and fungal infectionsto aging. Functional mitochondria are required for the propagation ofcancer stem cells. Inhibiting mitochondrial biogenesis in cancer cellsimpedes the propagation of those cells. Mitochondrial inhibitorstherefore represent a new class of anti-cancer therapeutics. Thesecompounds may also inhibit mitochondrial protein translation, andtherefore possess anti-microbial activity. As a result, mitochondrialinhibitors may function as broad-spectrum antibiotics that target bothbacteria and pathogenic yeast. Research has also showed thatmitochondrial inhibitors have anti-aging properties. This disclosureuses the term “mitoriboscins” to broadly describe thesemitochondrial-based therapeutic compounds having anti-cancer andantibiotic properties. Mitoriboscins at lower doses may be used totherapeutically target the aging process and to extend lifespan.

Novel mitochondrial inhibitors that target themitoribosome—mitoriboscins—may be identified through a convergentapproach of virtual high-throughput screening followed by in vivovalidation for mitochondrial inhibition. FIG. 2 is an overview ofmethods for identifying mitochondrial inhibitors by using in silico drugscreening and phenotypic drug screening disclosed herein. All or aportion of the three-dimensional structure of the mammalianmitochondrial ribosome (mitoribosome) may be used in step S101 toidentify novel compounds that bind to the mitoribosome through virtualhigh-throughput screening (vHTS) (i.e., in silico drug screening). Thescreening may be performed across a library of molecules. For instance,during initial investigations the inventors screened a collection of45,000 small molecule compounds for compounds expected to bind anywhereto the known large subunit of the large mitoribosome (39S), which is amulti-subunit complex with more than 50 subunits. Initial vHTS may usevarious screening programs, such as the eHiTS screening program, toidentify a subset of compounds having a strong binding affinity toeither the large or small subunit of the mammalian mitoribosome. Forexample, the inventors used eHiTS to identify the top 5,000 rankedcompounds from an initial library, based on predicted binding affinityto the large subunit (39S) of the mammalian mitoribosome. eHiTS is ascreening method that systematically covers the part of theconformational and positional search space that avoids severe stericclashes, producing highly accurate docking poses at a speed that iswell-suited for virtual high-throughput screening.

It should be appreciated that those skilled in the art may select ordevelop methods for identifying a subset of compounds having a desiredbinding affinity. To efficiently perform the docking, a series of clipfiles may be prepared corresponding to the entire protein structure andeach compound docked sequentially at each of the clip files. Consensusscoring of the top compounds may be carried out using AutoDock 4.2,based on the same general binding site for each compound predicted fromthe eHiTS screen. Further analysis of predicted binding affinity andvisual inspection may be carried out using a number of methods,including for example a de novo design program such as SPROUT. See Lawet al., J Mol Struct. 666: 651-657 (2003), which is incorporated byreference in its entirety, for information about SPROUT. Depending onthe initial library size and results, a number of compounds may beselected for phenotypic drug screening. For example, the inventorsselected 880 compounds that performed well in these analysis steps forphenotypic drug screening at step S103.

Phenotypic drug screening S103 may be accomplished by testing themitochondrial inhibition of selected compounds in a selected cell line.For example, ATP depletion assays may be used. The inventors tested theselected 880 compounds on their ability to functionally induceATP-depletion in MCF7 human breast cancer cells. Approximately 85% ofcellular ATP is normally generated by OXPHOS in mitochondria, soATP-depletion is a surrogate marker for mitochondrial inhibition. Itshould be appreciated that those skilled in the art may employ othersurrogates for mitochondrial inhibition. However, for the ATP-depletionassay inventors employed, MCF7 cells (6,000 cells/well) were plated intoblack clear-bottom 96-well plates and incubated overnight beforetreatment. The 880 compounds identified by vHTS were applied to theplated MCF7 cells at a concentration of 50 μM and were screened for ATPdepletion. Compounds showing ATP-depletion effects were subsequentlyre-screened at lower concentrations (25 μM and 10 μM) to identify thetop 10 compounds that most potently induce ATP-depletion. Compounds weretested after 72 hours of incubation and experiments were performed induplicate. After treatment, media was aspirated from the wells andplates were washed with warm phosphate-buffered saline (PBS)supplemented with Ca²⁺ and Mg²⁺. Then, cells were incubated with aHoechst 33342 (Sigma) staining solution (10 μg/ml) for 30 min and washedwith PBS to estimate cell viability. Fluorescence was read with a platereader using excitation/emission wavelengths at 355/460-nm. Then, theCellTiter-Glo luminescent assay (Promega) was performed to measuremetabolic activity (ATP content) in the very same wells that weretreated with a given compound. Assays were performed according to themanufacturer's protocol. Fluorescence intensity (Hoechst staining) andluminescence intensity (ATP content) were normalized to vehicle-alonetreated controls and were displayed as percent control for comparison.The results of this ATP depletion study are shown in FIG. 3. FIG. 3shows that all ten test compounds significantly depleted ATP levels inviable cells. It should be appreciated that those of skill in the artmay choose to employ the same or similar ATP-depletion assays, modifysuch assays, or may replace the ATP-depletion assay with anothermethodology for screening selected compounds for mitochondrialinhibition (e.g., oxygen consumption assays).

The present approach includes methods of confirming cell viability.Persons of skill in the art may select one or more methods forconfirming cell viability suitable for the particular embodiment. Theinventors initially used the Sulphorhodamine (SRB) assay, which is basedon the measurement of cellular protein content. After treatment for 72hours in 96-well plates, cells were fixed with 10% trichloroacetic acid(TCA) for 1 hour in the cold room, and were dried overnight at roomtemperature. Then, cells were incubated with SRB for 15 min, washedtwice with 1% acetic acid, and air dried for at least 1 hour. Finally,the protein-bound dye was dissolved in a 10 mM Tris, pH 8.8 solution andread using the plate reader at 540-nm. Using the SRB assay, theinventors selected only the compounds depleting ATP levels withoutprominent cytotoxicity for further analysis. Prominent cytotoxicity wasdefined as fewer than 30% of cells still on the plate. Of course,embodiments employing other cell viability confirmation methodology mayselect compounds for further analysis based on other considerations asmay be known in the art.

The present approach further involves methods of functional validationat step S105, during which a compound's function as a mitochondrialinhibitor may be confirmed. A number of methods may be used forfunctional validation, including, for example, metabolic flux analysis,mammosphere assays, viability assays, and antibiotic (anti-bacterialand/or anti-fungal) activity. For example, the inventors determinedextracellular acidification rates (ECAR) and real-time oxygenconsumption rates (OCR) for MCF7 cells using the Seahorse ExtracellularFlux (XF96) analyzer (Seahorse Bioscience, MA, USA). MCF7 cells weremaintained in DMEM supplemented with 10% FBS (fetal bovine serum), 2 mMGlutaMAX, and 1% Pen-Strep. 5,000 cells per well were seeded intoXF96-well cell culture plates, and incubated overnight at 37° C. in a 5%CO₂ humidified atmosphere. After 24 hours, cells were treated withselected compounds showing ATP-depletion without prominent cytotoxicityat various concentrations (or vehicle alone). After 72 hours oftreatment, cells were washed in pre-warmed XF assay media (for OCRmeasurement, XF assay media was supplemented with 10 mM glucose, 1 mMPyruvate, 2 mM L-glutamine and adjusted at pH 7.4). Cells weremaintained in 175 μL/well of XF assay media at 37° C. in a non-CO₂incubator for 1 hour. During incubation, 25 μL of 80 mM glucose, 9 μMoligomycin, 1M 2-deoxyglucose (for ECAR measurement) and 25 μL of 10 μMoligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCRmeasurement) in XF assay media was loaded into the injection ports ofthe XFe-96 sensor cartridge. During the experiment, the instrumentinjected these inhibitors into the wells at a given time point, whileECAR/OCR was measured continuously. ECAR and OCR measurements werenormalized by protein content (using the Sulphorhodamine B assay). Datasets were analyzed by XFe-96 software, using one-way ANOVA and Student'st-test calculations. All experiments were performed in triplicate, andresults validated the mitochondrial inhibition effects of mitoriboscincompounds described herein. It should be appreciated that numerousmethods are known for functional validation, and that persons of skillin the art may select one or more depending on the validation needs(e.g., other assays that measure or approximate mitochondrial function).

In summary, the present approach provides methods of identifyingpotential mitochondrial inhibitors and mitoriboscins using in silicodrug screening and phenotypic drug screening. Novel compounds identifiedusing this methodology may be tested for anti-cancer activity (e.g., theability to inhibit mammosphere formation and cell migration) and may befurther tested on distinct bacterial and/or yeast strains to investigateanti-microbial activity. FIG. 2 summarizes the general methods accordingto embodiments of the present approach, but it should be appreciatedthat those of skill in the art may deviate from the specific examplesdisclosed herein without departing from the present approach.

The present approach has led to the identification of categories ofmitochondrial-inhibiting compounds—and in particular mitoriboscins—thathave anti-cancer, anti-microbial, and anti-aging properties. Based onthe inventors' initial screening and validation, the compoundsidentified in FIG. 4 have anti-cancer, anti-microbial, and anti-agingproperties. These unique mitoriboscins are therefore candidates forclinical trial. It should be appreciated that the mitoriboscinsidentified in FIG. 4 are not exhaustive, but are merely those that havebeen identified thus far using the novel methodology set forth herein.

Four groups of mitoriboscins have been identified, as shown in FIGS.4A-4D and 12. The mitoriboscin groups shown in FIG. 12,mitoribocyclines, mitoribomycins, mitoribosporins, and mitoribofloxins,may be selected for use as anti-cancer, antibiotic, and/or anti-agingtherapeutics. It should be appreciated by those skilled in the art thatthe therapeutically-effective amount of each compound, for a particulartherapy, depends on a multitude of factors. In some embodiments,combinations of compounds from one or more mitoriboscin groups may beused as anti-cancer, antibiotic, and/or anti-aging therapeutics.

In some embodiments, the mitoriboscin compound comprises the generalformula or salts thereof:

wherein each R may be the same or different and is selected from thegroup consisting of hydrogen, carbon, nitrogen, sulfur, oxygen,flourine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes, arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, benzoic acid-basedderivatives, and one or more mitochondrial targeting signals. Forclarification, mitochondrial targeting signals are defined as anychemical or peptide entity that increases the efficiency of targetingthe attached molecule to the mitochondria. Such modification would beexpected to increase the potency and effectiveness of a mitoriboscin.Thus, R may be any mitochondrial targeting signal (peptide or chemical),including cationic compounds, such as tri-phenyl-phosphonium (TPP), aguanidinium-based moiety and/or choline esters, among others.

In some embodiments, the mitoriboscin compound comprises the generalformula or salts thereof:

wherein each R may be the same or different and is selected from thegroup consisting of hydrogen, carbon, nitrogen, sulfur, oxygen,fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes,alkane-based derivatives, alkenes, cyclic alkenes, alkene-basedderivatives, alkynes, alkyne-based derivative, ketones, ketone-basedderivatives, aldehydes, aldehyde-based derivatives, carboxylic acids,carboxylic acid-based derivatives, ethers, ether-based derivatives,esters and ester-based derivatives, amines, amino-based derivatives,amides, amide-based derivatives, monocyclic or polycyclic arene,heteroarenes, arene-based derivatives, heteroarene-based derivatives,phenols, phenol-based derivatives, benzoic acid, benzoic acid-basedderivatives, and one or more mitochondrial targeting signals.

In some embodiments, the mitoriboscin compound comprises the generalformula or salts thereof:

wherein R is selected from the group consisting of hydrogen, carbon,nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl,alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclicalkenes, alkene-based derivatives, alkynes, alkyne-based derivative,ketones, ketone-based derivatives, aldehydes, aldehyde-basedderivatives, carboxylic acids, carboxylic acid-based derivatives,ethers, ether-based derivatives, esters and ester-based derivatives,amines, amino-based derivatives, amides, amide-based derivatives,monocyclic or polycyclic arene, heteroarenes, arene-based derivatives,heteroarene-based derivatives, phenols, phenol-based derivatives,benzoic acid, benzoic acid-based derivatives, and one or moremitochondrial targeting signals.

In some embodiments, the mitoriboscin compound comprises the generalformula or salts thereof:

The specific mitoriboscins of the formulas shown in FIG. 4 are shown asspecific examples of the groups of mitoriboscins identified in FIG. 12.It should be appreciated that the mitoriboscins may be selected fortherapeutic use individually, or in combination with more than onespecific mitoriboscin, and/or with other substances to enhance theefficacy of other therapeutics. The therapeutics may be used in the formof usual pharmaceutical compositions which may be prepared using one ormore known methods. For example, a pharmaceutical composition may beprepared by using diluents or excipients such as, for example, one ormore fillers, bulking agents, binders, wetting agents, disintegratingagents, surface active agents, lubricants, and the like as are known inthe art. Various types of administration unit forms can be selecteddepending on the therapeutic purpose(s). Examples of forms forpharmaceutical compositions include, but are not limited to, tablets,pills, powders, liquids, suspensions, emulsions, granules, capsules,suppositories, injection preparations (solutions and suspensions),topical creams, and other forms as may be known in the art. For thepurpose of shaping a pharmaceutical composition in the form of tablets,any excipients which are known may be used, for example carriers such aslactose, white sugar, sodium chloride, glucose, urea, starch, calciumcarbonate, kaolin, cyclodextrins, crystalline cellulose, silicic acidand the like; binders such as water, ethanol, propanol, simple syrup,glucose solutions, starch solutions, gelatin solutions, carboxymethylcellulose, shelac, methyl cellulose, potassium phosphate,polyvinylpyrrolidone, etc. Additionally, disintegrating agents such asdried starch, sodium alginate, agar powder, laminalia powder, sodiumhydrogen carbonate, calcium carbonate, fatty acid esters ofpolyoxyethylene sorbitan, sodium laurylsulfate, monoglyceride of stearicacid, starch, lactose, etc., may be used. Disintegration inhibitors suchas white sugar, stearin, coconut butter, hydrogenated oils; absorptionaccelerators such as quaternary ammonium base, sodium laurylsulfate,etc., may be used. Wetting agents such as glycerin, starch, and othersknown in the art may be used. Adsorbing agents such as, for example,starch, lactose, kaolin, bentonite, colloidal silicic acid, etc., may beused. Lubricants such as purified talc, stearates, boric acid powder,polyethylene glycol, etc., may be used. If tablets are desired, they canbe further coated with the usual coating materials to make the tabletsas sugar coated tablets, gelatin film coated tablets, tablets coatedwith enteric coatings, tablets coated with films, double layered tabletsand multi-layered tablets. Pharmaceutical compositions adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, foams, sprays,aerosols, or oils. Such pharmaceutical compositions may includeconventional additives which include, but are not limited to,preservatives, solvents to assist drug penetration, co-solvents,emollients, propellants, viscosity modifying agents (gelling agents),surfactants and carriers.

The present approach involves methods of testing compounds, and inparticular mitoriboscins, for anti-cancer properties. As discussedabove, vHTS and computational chemistry may be used to identifycandidate mitochondrial inhibitors. Those candidates may be tested forspecific anti-cancer properties. For examples, the inventors comparedseven candidate compounds in parallel for their ability to inhibitmammosphere formation in MCF7 cells. FIG. 5 illustrates how five of theseven compounds tested significantly inhibited mammosphere formation ata concentration of 5 μM. For example, 23/G4 (Group 1) reducedmammosphere formation by 50% at this concentration. Similarly, 24/F9(Group 2) and 24/D4 (Group 3), both reduced mammosphere formation by˜90%.

Based on this analysis, the inventors assessed the functional effects ofthe three candidates on overall viability in MCF7 cell monolayers andnormal human fibroblasts (hTERT-BJ1 cells) (FIG. 6). 23/G4 (Group 1)reduced the viability of MCF7 cells by 70% at a concentration of 5 μM.However, 23/G4 had no effect on the viability of hTERT-BJ1 cells, whentested at the same concentration. Thus, it is possible to identifycompounds, such as 23/G4, that preferentially target CSCs and “bulk”cancer cells, but not normal fibroblasts. Those of skill in the art maydetermine the preferential targeting of candidate mitoriboscinsemploying the same method or other methods known in the art.

The present approach involves methods of function validation ofmitoriboscin compounds. For example, the inventors assessed functionalvalidation of three candidates using the Seahorse Analyzer, whichquantitatively measures oxygen consumption rate (OCR) and extracellularacidification rate (ECAR). OCR is a surrogate marker for OXPHOS and ECARis a surrogate marker for glycolysis and L-lactate production.

The inventors' results demonstrated that 23/G4 (Group 1), 24/F9 (Group2) and 24/D4 (Group 3) all dose-dependently inhibited mitochondrialoxygen-consumption in MCF7 cells, with 23/G4 being the most potent(FIGS. 7, 8 and 9). 23/G4 reduced ATP levels by >50% at a concentrationof only 500 nM. In addition, 23/G4 reduced ATP levels by ˜75% at 2.5 μM(FIG. 7). Remarkably, treatment with 23/G4, at the same concentrations,had little or no effect on the overall cell viability of MCF7 monolayers(FIG. 6). Therefore, 23/G4 very effectively depleted ATP levels, withoutshowing significant cytotoxicity.

The data demonstrated that 23/G4 induced an increase in glycolysis ratesby more than 1.5-fold, while 24/F9 and 24/D4 both suppressed glycolysis.This could explain why 24/F9 and 24/D4 were more potent than 23/G4 inthe mammosphere assay, where 24/F9 and 24/D4 both reduced mammosphereformation by ˜90% at a concentration of 5 μM (FIG. 5). The rank orderpotency of the top 10 hits for their ability to reduce i) maximalrespiration and ii) ATP production is shown in FIG. 10. Note that thetop 6 compounds in this regard were 23/G4, 25/B3, 24/H9, 24/F9, 23/E9and 24/H6, with 23/G4 being the most potent, yielding a greater than 75%reduction in ATP levels at 5 μM.

As EMT and cell invasion are phenotypic features associated with“stemness” and distant metastasis, the effects of these compounds on theability of another more aggressive breast cancer cell line, MDA-MB-231,to undergo cell migration were evaluated. FIG. 11 shows that 23/G4,24/D4 and 24/F9 all inhibited cell migration by more than 70%, at aconcentration of 2.5 μM.

The present approach allows for testing compounds for anti-cancerproperties by considering compound effects on mammosphere formation andcell migration. Using the methods disclosed herein, 23/G4 (Group 1)appears to be a promising new lead compound, as it is more selective attargeting CSCs and cancer cells, while sparing normal cells (FIG. 6).23/G4 is the most potent hit compound that effectively reducesmitochondrial ATP levels and induces glycolysis. It should beappreciated that those skilled in the art may use other methods known inthe art for assessing a candidate mitochondrial inhibitor's effects on aparticular cell line without departing from the present approach. Itshould also be appreciated that those skilled in the art may assess acandidate mitochondrial inhibitor's effects on other cancer types, asthe inhibitors target cancer stem cells (CSCs). CSCs show conservedfeatures across most cancer types. Antibiotics such as doxycycline anderythromycin, which bind to mitochondrial ribosomes as an off-targeteffect, show efficacy in twelve different cell lines, representing eightdifferent cancer types. These include: ductal carcinoma in situ (DCIS),breast, ovarian, pancreatic, lung carcinomas, as well as melanoma andglioblastoma.

FIG. 1 depicts the evolution of aerobic bacteria into mitochondrialorganelles over millions of years of symbiosis and adaptation. In viewof this evolutionary history, compounds that target mitochondrialprotein translation in cancer cells may also possess anti-microbialactivity. The present approach provides methods of testing compounds foranti-microbial activity, as well as novel compounds havinganti-microbial activity. To demonstrate that mitochondrial inhibitorsfunction as broad-spectrum antibiotics, the inventors tested theanti-microbial activity of the top three compounds (24/F9, 24/D4 and23/G4) against two gram-positive bacterial strains (Staph. aureus andStrep. pyogenes) three gram-negative bacterial strains (E. coli, P.aeruginosa, K. pneumoniae), and the pathogenic yeast strain C. albicans.

The antimicrobial effects of a candidate mitochondria inhibitor may beevaluated using the Kirby-Bauer disc-diffusion method, performedaccording to the Clinical and Laboratory Standards Institute (CLSI)guidelines, and results are interpreted using CLSI breakpoints.Antibiotics disks against gram (+ve) and gram (−ve) bacteria (fromOxoid™) may be used as positive controls. The inventors assessed theantibiotic effects of certain mitoriboscins described herein based onthe following methodology. Compounds 24/D4, 24/F9, and 23/G4 identifiedherein were prepared by dissolving them in dimethyl sulfoxide (DMSO,from Sigma/Aldrich Company; St. Louis, Mo., USA) and were utilized toimpregnate the Blank Antimicrobial Susceptibility Disks (Oxoid™).Overnight cultures of bacteria tested were adjusted to a turbidity of0.5 McFarland standards (10⁶ CFU/ml) before inoculation onto agar plateswith sterile cotton swabs. A cotton swab dipped in the cell culture wasstreaked onto an agar plate surface in such a way as to obtain a uniformlayer of bacteria across the whole surface. After 10-15 minutes, theantibiotics disks or novel compounds disks were placed on the inoculatedsurface of the agar plates; then, all agar plates were incubated at 37°C. overnight. The diameters of inhibition were measured andsusceptibility was expressed in terms of resistance (R), moderatesusceptibility (I) and susceptibility (S). Agar plates inoculated withbacteria tested with impregnated DMSO disks were used as controls. Theresult obtained on a single bacteria strain was confirmed by Sensi testgram-positive and Sensi test-gram-negative kits (Liofilchem S.R.L.).Disc-diffusion susceptibility tests were performed in triplicate andrepeated three times independently.

The minimal inhibitory concentration (MIC) of the antibacterialcompounds may be determined using the broth dilution method, accordingto CLSI guidelines. Test compound solutions (or antibiotic solutionsused as positive controls) were diluted, serially, with MHB medium.Then, the suspensions of the microorganisms, prepared from overnightcultures of bacteria in the MHB medium, at a concentration of 10⁶CFU/ml, were added to each dilution in a 1:1 ratio. McFarland standardswere used as a reference to adjust the turbidity of microorganismsuspensions. Growth (or lack thereof) of the microorganisms wasdetermined after incubation for 24 hours at 37° C. by turbidimetry(wavelength of 600 nm). MIC 50 and MIC 99 are defined as the minimuminhibitory concentration of the compound required for 50% and 99%inhibition of bacterial growth. The negative control tubes did notcontain bacterial inoculum and the positive control tubes contained onlyDMSO. The susceptibility test by measurement of MIC was performed intriplicate and repeated three times independently. Statisticalsignificance was determined using the Student's t-test and values ofless than 0.05 were considered significant.

TABLE 1 Gram-Positive Bacterial Antibiotic Sensitivity. Staph. aureusStrept. pyogenes Susceptibility Testing Gram-positive ATCC 25923 ATCC19615 ANTIBIOTIC/ CONTENT EVALUATION INHIBITOR μg S I R S I RCiprofloxacin 4 X X Rifampicin 4 X X 23/G4 8 X X Gentamicin 8 X XTobramycin 8 X X Levofloxacin 8 X X Pefloxacin 8 X X Azithromycin 8 X XClarithromycin 8 X X Erithromycin 8 X X Miokamycin 8 X X Roxitromycin 8X X Co-trimoxazole 8 X X Amoxicillin/ 8/4 X X Clavulanic acidPiperacillin 16 X X 24/D4 32 X X Netilmicin 32 X X Cefaclor 32 X XCefixime 32 X X Cefonicid 32 X X Ceftazidime 32 X X Cefuroxime 32 X XAmpicillin/Sulbactam 32/16 X X 24/F9 64 X X Ceftriaxone 64 X XFosfomycin 200 X X

Table 1 summarizes gram-positive anti-bacterial activity ofmitoriboscins compounds 24/F9, 24D4, and 23/G4 compared to knownantibiotics and across two gram-positive bacterial strains (Staph.aureus and Strep. pyogenes). The column label S identifies sensitivity,I identifies intermediate, and R identifies resistant. Tables 1 and 2illustrate how all five bacterial strains tested are sensitive to themitoriboscin compounds (24/F9, 24/D4 and 23/G4). No growth inhibitionwas seen in the control (DMSO).

TABLE 2 Gram-Negative Bacterial Antibiotic Sensitivity. E. coli P.auriginosa K. pneumoniae Susceptibility Testing Gram-negative ATCC 25922ATCC 27853 ATCC 13883 ANTIBIOTIC/ CONTENT EVALUATION INHIBITOR μg S I RS I R S I R Ciprofloxacin 4 X X X Rifampicin 4 X X X Gentamicin 8 X X XTobramycin 8 X X X Lomefloxacin 8 X X X Levofloxacin 8 X X X Pefloxacin8 X X X Co-trimoxazole 8 X X X 24/D4 32 X X X 23/G4 32 X X X Amikacin 32X X X Ceftazidime 32 X X X Cefuroxime 32 X X X Nalidixic acid 32 X X XTeicoplanin 32 X X X Aztreonam 32 X X X Amoxicillin/Clavulanic acid32/16 X X X Ampicillin/Sulbactam 32/16 X X X Cefotaxime 64 X X X 24/F964 X X X Cefoperazone 64 X X X Cefotaxime 64 X X X Ceftriaxone 64 X X XNitrofurantoin 128 X X X Piperacillin/Tazobactam 128/4  X X XTicarcillin/Clavulanic acid 128/4  X X X Fosfomycin 200 X X X

Table 2 summarizes the anti-bacterial activity of mitoriboscin compounds24/F9, 24/D4 and 23/G4, compared to known antibiotics, across threedifferent gram-negative bacterial strains (E. coli, P. aeruginosa, K.pneumoniae). The column label S identifies sensitivity, I identifiesintermediate, and R identifies resistant.

In order to determine the minimal inhibitory concentration (MIC) for24/F9, 24/D4 and 23/G4, the broth dilution method may be performed.Using this method, the MIC determination results demonstrated agreementwith the disc-diffusion susceptibility test.

TABLE 3 Minimum Inhibitory Concentrations (MIC): Bacterial Strains andPathogenic Yeast Minimum inhibitory concentration (compare with commonantibiotics) MIC μg/ml E. coli P. auriginosa K. pneumoniae Staph. aureusStrept. pyogenes C. albicans ANTIBIOTIC/ ATCC 25922 ATCC 27853 ATCC13883 ATCC 25923 ATCC 19615 ATCC 13883 INHIBITOR 50% 99% 50% 99% 50% 99%50% 99% 50% 99% 50% 99% 24/D4 16 32 16 32 16 32 16 32 16 32 — >6423/G4 >16 >32 16 32 16 32 4 8 4 8 8   16 24/F9 32 64 >32 >64 32 64 32 6432 64 — >64 DOXYCYCLINE 0.5 2 1 2 2 4 0.5 1 0.5 1 — >64 LINEZOLID128 >128 64 256 128 256 1 2 1 2 — — AMOXICILLIN 16 >32 128 256 32 >64 48 4 8 — — MICONAZOLE — — — — — — — — — — 0.5 1

Table 3 shows the MIC determination results obtained as compared toknown antibiotics, against the tested bacterial strains and C. albicans.Compound 23/G4 shows the greatest broad-spectrum activity and potency,as compared with compounds 24/F9 and 24/D4.

TABLE 4 Minimum Inhibitory Concentrations (MIC): MRSA v. MSSA MIC μg/mlMinimum inhibitory concentration MRSA MSSA (compare with commonantibiotics) ATCC 43300 ATCC 25923 ANTIBIOTIC/INHIBITOR 50% 99% 50% 99%24/D4 16 >32 16 32 23/G4 16 >32 4 8 24/F9 64 >64 32 64AMOXICILLIN >64 >64 4 8

Table 4 shows that Methicillin-resistant Staphylococcus aureus (MRSA) isalso sensitive to 23/G4 and 24/D4. It was confirmed that this strain ofMRSA was indeed resistant to amoxicillin, as predicted. This resultshows it may be possible to use this new drug discovery strategyemploying human cancer cells to isolate new antibiotics that can targetdrug-resistant bacteria, such as MRSA.

The data demonstrates that the mitoriboscins identified by the presentapproach have anti-cancer, anti-bacterial properties, and are suitablefor pharmaceutical compositions.

The inventors have shown that compounds inducing acute ATP depletion incancer cells can sensitize those cells to radiation, ultraviolet light,chemotherapeutic agents, natural substances, and/or caloric restriction.Mitoriboscins, as discussed herein, have demonstrated ATP-depletioneffects. Based on these preliminary results, mitoriboscins may also beused as radiosensitizers and/or photo-sensitizers. Use asradiosensitizers and/or photo-sensitizers may be in combination withother treatment vectors, including but not limited to other cancertreatment methods as may be known in the art, and cancer treatmentthrough inhibiting mitochondrial biogenesis as disclosed herein.Similarly, mitoriboscins may be used to functionally sensitize bulkcancer cells and cancer stem cells to chemotherapeutic agents,pharmaceuticals, and/or other natural substances, such as dietarysupplements and caloric restriction.

In addition to anti-cancer and anti-biotic behavior, the mitochondrialinhibitors that can be identified by the present approach have thepotential to slow the mammalian aging process. Genetic inhibition ofmitochondrial protein translation has been shown to have beneficialside-effects, and in particular the side effect of slowing of the agingprocess and increased lifespan in model organisms. Lower steady-statelevels of Mrps5 (a mitoribosomal protein) is strongly functionallycorrelated with longer murine lifespan, resulting in a significantincrease of ˜250 days. In addition, selective knock-down of Mrps5 in C.elegans dramatically increases lifespan. Mrps5 knock-down worms showsignificant decreases in mitochondrial respiration and ATP production.Similarly, knock-down of the worm homologs of mitochondrial complex I,III, IV and V, as well as several TCA cycle enzymes, all robustlyextended lifespan, further implicating reduced OXPHOS activity and lowerATP levels as the mechanism. Finally, pharmacological inhibition ofmitochondrial biogenesis (using the off-target effects of doxycycline),also significantly increases lifespan in C. elegans. Thus, lower dosesof the mitoriboscins may be used to therapeutically target the agingprocess and to extend lifespan.

Mitoriboscins may also be used to reverse drug resistance in cancercells. Drug resistance is thought to be based, at least in part, onincreased mitochondrial function in cancer cells. In particular, cancercells demonstrating resistance to endocrine therapies, such astamoxifen, are expected to have increased mitochondrial function.Mitoriboscins inhibit mitochondrial function, and therefore may beuseful in reducing and, in some cases reversing, drug resistance incancer cells.

Mitoriboscins may also be used as male contraceptives and/or asspermistatic or sperm-immobilizing agents. The human sperm cell consistsof a head and a flagellum. The flagellum includes a neck, middle piece,and tail. The middle piece typically has 10-14 spirals of mitochondriasurrounding the axial filament in the cytoplasm. These mitochondriaprovide motility to the sperm and thus are often referred to as the“powerhouse of the sperm.” Mitoriboscins inhibit mitochondrial functionand therefore may be useful in immobilizing sperm cells to preventconception.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. The invention includes numerousalternatives, modifications, and equivalents as will become apparentfrom consideration of the following detailed description.

It will be understood that although the terms “first,” “second,”“third,” “a),” “b),” and “c),” etc. may be used herein to describevarious elements of the invention should not be limited by these terms.These terms are only used to distinguish one element of the inventionfrom another. Thus, a first element discussed below could be termed aelement aspect, and similarly, a third without departing from theteachings of the present invention. Thus, the terms “first,” “second,”“third,” “a),” “b),” and “c),” etc. are not intended to necessarilyconvey a sequence or other hierarchy to the associated elements but areused for identification purposes only. The sequence of operations (orsteps) is not limited to the order presented in the claims.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. Thus, the term“consisting essentially of” as used herein should not be interpreted asequivalent to “comprising.”

The term “about,” as used herein when referring to a measurable value,such as, for example, an amount or concentration and the like, is meantto encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount. A range provided herein for a measurable value mayinclude any other range and/or individual value therein.

Having thus described certain embodiments of the present invention, itis to be understood that the invention defined by the appended claims isnot to be limited by particular details set forth in the abovedescription as many apparent variations thereof are possible withoutdeparting from the spirit or scope thereof as hereinafter claimed.

What is claimed is:
 1. A mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceutical composition comprising a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 3. A method of treating cancer comprising administering to a patient in need thereof of a pharmaceutically effective amount of a mitoriboscin comprising the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 4. A method of sensitizing cancer cells to a chemotherapeutic agent, the method comprising administering to a patient in need thereof of a pharmaceutically effective amount of a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 5. A method of sensitizing cancer cells to caloric restriction, the method comprising administering to a patient in need thereof of a pharmaceutically effective amount of a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 6. A method of sensitizing cancer cells to radiation therapy, the method comprising administering to a patient in need thereof of a pharmaceutically effective amount of a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 7. A method of sensitizing cancer cells to photodynamic therapy, the method comprising administering to a patient in need thereof of a pharmaceutically effective amount of a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 8. A method of killing microorganisms, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of a mitoriboscin of the formula

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 